Neural network reconstruction on sliced acellular brains for brain organoids Tsuyoshi Kimura1, Naoko Nakamura1, Yoshihide Hashimoto1, Aoi Odawara2, Ikuro Suzuki2, Akio Kishida1 1 Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2 Department of Electronics, Tohoku Institute of Technology.

Statement of Purpose: Organoid models are expected in Results: Rat brains were decellularized by using various many basic biology and clinical applications [1]. In methods, HHP, SDS and SDC. The SDC and SDS general, organoid have been generated from both decellularized brain was swollen. The remaining of cells pluripotent stem cell and adult stem cells by mimicking was observed in the decellularized brain by their detergent the physiological condition. Recently, several methods (data not shown). For the HHP decellularized bioengineering approaches that were developed in the brain, the removal of cell was achieved with maintaining fields, stem cell niche and tissue engineering, have the structure of brain (Fig 2A, 2B). The rat embryonic become available to generate organoids [2]. On the other hippocampal cells, neural and glia cells, were seeded on hand, decellularized tissues/organs are biocompatible the HHP decellularized brain. The cells were stained by scaffold for regenerative medicine. Recently, in vitro re- Calcein-AM. It was observed that cells were adhered on construction of tissues/organs using them is attempted the HHP decellularized brain (Fig 2C). For fluorescent because the ECM of decellularized tissues/organs affects immunostaining using anti-beta-tubulin antibody (neural on the cellular behaver and organization. We also have cells) and anti-GFAP antibody (glia cells), both of neural developed decellularized tissues, such as aorta, cornea and glia cells were observed on the brain. Especially, in and bone marrow, using a high hydrostatic pressure the region of cerebral cortex, the many neural cells were (HHP) technology [3]. The HHP decellularization method adhered and the network of neural cells was also formed can decellularize tissue remaining the extracellular matrix (Fig2D). (ECM) structure compared with chemical decellularization methods using detergents. We recently focus on the influence of the ECM structure of A B D decellularized tissue on cellular behavior on/in it. We I II hypothesize that the ECM structure of HHP decellularized tissue is preserved as well as that of native tissue and allows the essential nature and behavior of cell and leads C to generate organoid. In this study, we prepared a decellularized brain using the HHP method and investigated neural cell behavior on the HHP decellularized brain in vitro. Green:&Neuron,&Red:& Astrocyte,&Blue:&Nucleus Fig.2 Reconstructed brain cultured with rat embryonic Methods: Brain was harvested from Wistar rat (8weeks). hippocampal cells. Brains were decellularized by sodium deoxycholate (SDC), sodium dodecyl sulfate (SDS) and HHP methods. Conclusions: In the present study, rat brains were For SDC and SDS decellularization, brain was sliced decellularized by SDC, SDS and HHP methods. For SDC using a brain slicer and treated with their detergent and SDS decellularization methods, it was difficult to solutions for 24 hours. For HHP decellularization, the remain the ECM structure of brain. For the HHP whole brain was pressurized at 10,000 atm for 10 min. decellularization method, the removal of cells and The pressurized brain was sliced and washed with saline maintenance of ECM structure were confirmed. Also, rat containing DNase I and PBS. Rat embryonic hippocampal embryonic hippocampal cells could be cultured on the cells were seeded on the HHP decellularized brains. After HHP decellularized brain. So, we have successfully several weeks, they were subjected to fluorescent developed the decellularized brain using HHP technology immunostaining and observed with a fluorescent as a brain organoid scaffold. microscope. (Fig 1) References: 1. N. Barker, et al, Nat Cell Biol, 18, (2016) 246.

2. O. Levy, et al., Cell Stem Cell, 18 (2016) 25. 3. Y. Hashimoto, et al, Biomaterials. 14 (2010) 3941.

Fig.1 Preparation and recellularization of decellularized brains.